Method for secretory production of glycoprotein having human-type sugar chain using plant cell

ABSTRACT

A method for the secretory production of a glycoprotein having a human-type sugar chain, comprising a step of introducing a gene of an enzyme capable of performing a transfer reaction of a galactose residue to a non-reducing terminal acetylglucosamine residue, and a gene of heterologous glycoprotein, to obtain a transformed plant cell, a step of culturing the plant cell, and a step of recovering the culture medium of the plant cell.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/717,956, filed Mar. 14, 2007, which is a continuation of U.S.application Ser. No. 10/466,941, filed on Jul. 18, 2003, which is anational stage filing under 35 U.S.C. 371 of International ApplicationPCT/JP2002/00361, filed Jan. 18, 2002, which claims priority fromJapanese Application No. 2001-12519, filed Jan. 19, 2001, the entirecontents of each of which are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to a method for the secretory productionof a heterologous glycoprotein having a human-type sugar chain using aplant cell, a plant cell capable of secreting this glycoprotein, and aglycoprotein having a human-type sugar chain secreted by this plantcell.

BACKGROUND ART

Production of extraneous proteins using plant cultured cells isproceeding. For example, attempts are being made to produce thefollowing proteins useful for humans using tobacco cultured cell:

GM-CSF (see, E. A. James, C. Wang, Z. Wang, R. Reeves, J. H. Shin, N. S.Magnuson and J. M. Lee, “Production and Characterization of BiologicallyActive Human GM-CSF Secreted by Genetically Modified Plant Cells”,Protein Expr. Purif., 19, 131-138 (2000)), IL-2 and IL-4 (see, N. S.Magnuson, P. M. Linzmaier, R. Reeves, G. An, K. HayGlass and J. M. Lee,“Secretion of Biologically Active Human Interleukin-2 and Interleukin-4from Genetically Modified Tobacco Cells in Suspension Culture”, ProteinExpr. Purif., 13, 45-52 (1998)), immunoglobulin (see, N. S. Magnuson, P.M. Linzmaier, J. W. Gao, R. Reeves, G. An and J. M. Lee, “EnhancedRecovery of a Secreted Mammalian Protein from Suspension Culture ofGenetically Modified Tobacco Cells”, Protein Expr. Purif 7, 220-228(1996)), erythropoietin (see, S. Matsumoto, A. Ishii, K. Ikura, M. Uedaand R. Sasaki, “Expression of Human Erythropoietin in Cultured TobaccoCells”, Biosci. Biotechnol., Biochem., 57, 1249-1252 (1993)), andα1-antitrypsin (see, M. Terashima, Y. Murai, M. Kawamura, S. Nakanishi,T. Stoltz, L. Chen, W. Drohan, R. L. Rodriguez and S. Katoh, “Productionof Functional Human α1-Antitrypsin by Plant Cell Culture”, Appl.Microbiol. Biotechnol., 52, 516-523 (1999)).

On other hand, it is reported that plant cultured cells secrete manyproteins or glycoproteins (see, A. Sturm, “Heterogeneity of the ComplexN-Linked Oligosaccharides at Specific Glycosylation Sites of TwoSecreted Carrot Glycoproteins”, Eur. J. Biochem., 199, 169-179 (1991);Y. Okushima, N. Koizumi, T. Kusano and H. Sano, “Secreted Proteins ofTobacco Cultured BY2 Cells: Identification of A New Member ofPathogenesis-Related Proteins”, Plant Mol. Biol., 42, 479-488 (2000);and Y. Okushima, N. Koizumi, T. Kusano and H. Sano, “Glycosylation andIts Adequate Processing is Critical for Protein Secretion in Tobacco BY2Cells”, J. Plant Physiolo., 154, 623-627 (1999)). Of these, in the caseof tobacco BY2 cultured cells, two kinds of peroxidases are purified andtheir genes have been cloned (see, H. Narita, Y. Asaka, K. Ikura; S.Matsumoto and R. Sasaki, “Isolation, Characterization and Expression ofCationic Peroxidase Isozymes Released into the Medium of CulturedTobacco Cells”, Eur. J. Biochem., 228, 855-862 (1995)). It is alsoreported that by adding polyvinylpyrrolidone (PVP) to the medium, theconcentration of protein secreted in the medium could be increased (see,N. S. Magnuson, P. M. Linzmaier, J. W. Gao, R. Reeves, G. An and J. M.Lee, “Enhanced Recovery of A Secreted Mammalian Protein from SuspensionCulture of Generically Modified Tobacco Cells”, Protein Expr. Purif., 7,220-228 (1996)) and by Y. Okushima et al., supra. (1999) that fromtobacco BY2 strain cultured cells, hundreds of proteins areextracellularly secreted. Among these, extracellular secretion of manyglycoproteins are confirmed because of their reaction with lectin(concanavalin A) which recognizes high mannose-type sugar chains (see,Y. Okushima, supra. (1999)).

With respect to these glycoproteins, in particular, immunoglobulin,interleukin and GM-CSF, produced within plant cells, a signal peptide ofeach glycoprotein itself is also recognized in the secretion mechanismwithin the plant cell, and is secreted in the culture solution (see, E.A. James et al., supra.; N. S. Magnuson et al., supra. (1998); and N. S.Magnuson et al., supra. (1996)). In any of these glycoproteins, it issuggested, the sugar chain participates in the determination ofhalf-life in blood, sensitivity to protease and stability. However, thesugar chain structures of recombinant proteins actually produced withinplant cells and purified have not been examined and these proteins arepresumed to have a plant-type sugar chain structure.

In the analysis of sugar chain structure, the secretion-type antibodymolecule sIgA, produced from tobacco plants is revealed to have aplant-type sugar chain (see, M. Cabanes-Macheteau, A. C.Fitchette-Laine, C. Loutelier-Bourhis, C. Lange, N. D. Vine, J. K. Ma,P. Lerouge and L. Faye, “N-Glycosylation of a Mouse IgG Expressed inTransgenic Tobacco Plants”, Glycobiology, 9, 365-372 (1999)).Furthermore, in the case where another antibody molecule is producedfrom the same tobacco plant body, the antibody protein produced withinthe cell is decomposed by the protease and is unstable (see, L. H.Stevens, G. M. Stoopen, I. J. Elbert, J. W. Molthoff, H. A. Bakker, A.Lommen, D. Bosch and W. Jordi, “Effect of Climate Conditions and PlantDevelopmental Stage on the Stability of Antibodies Expressed inTransgenic Tobacco”, Plant Physiol., 124, 173-182 (2000)). By theWestern method using an antiplant-type sugar chain antibody, addition ofa plant-type sugar chain to this antibody is confirmed. Although it isreported that the β1,4-linked galactose residue present in the sugarchain of an antibody molecule produced by human or mouse contributes tothe stabilization of antibody protein, this sugar residue is absent inthe antibody molecule produced by plant cells. Because of this, theantibodies produced by tobacco plants are considered to be prone todecomposition by the protease.

In the case where erythropoietin is produced by tobacco cultured cells,the biological activity is recognized in vitro, but the activity in vivois not detected (see, S. Matsumoto, K. Ikura, M. Ueda and R. Sasaki,“Characterization of a Human Glycoprotein (Erythropoietin) Produced inCultured Tobacco Cells”, Plant Mol. Biol., 27 1163-1172 (1995). This isconcluded to occur because erythropoietin of which sugar chain isconsidered to greatly participate in the biological activity, has alargely different sugar chain structure when produced by plant cells.

On the other hand, it is suggested that the plant-type sugar chain maybe an allergen in mammals including humans. That is, the sugar chainstructure peculiar to plants, such as β1,2-xylose and α1,3-fucose whichare not seen in glycoproteins of mammals, are reported to act as anallergen (see, K. Fotisch, F. Altmann, D. Haustein and S. Vieths,Involvement of Carbohydrate Epitopes in the IgE Response ofCelery-Allergic Patients, Int. Arch. Allergy Immunol., 120, 30-42(1999); I. B. Wilson, J. E. Harthill, N. P. Mullin, D. A. Ashford and F.Altmann, “Core α1,3-Fucose is a Key Part of the Epitope Recognized byAntibodies Reacting Against Plant N-Linked Oligosaccharides and isPresent in a wide Variety of Plant Extracts”, Glycobiology, 8, 651-661(1998); and R. van Ree, M. Cabanes-Macheteau, J. Akkerdaas, J. P.Milazzo, C. Loutelier-Bourhis, C. Rayon, M. Villalba, S. Koppelman, R.Aalberse, R. Rodriguez, L. Faye and P. Lerouge, “β(1,2)-Xylose andα(1,3)-Fucose Residues Have a Strong Contribution in IgE Binding toPlant Glycoallergens”, J. Biol. Chem., 275(15), 11451-11458 (Apr. 14,2000). Accordingly, proteins for medical uses must have a sugar chainstructure free of β1,2-xylose or α1,3-fucose.

DISCLOSURE OF THE INVENTION

The object of the present invention is to solve the above-describedproblems in conventional techniques and provide a method for thesecretory production, in plant cells, of a glycoprotein which is stable,maintains its original physiological activity and is not an allergen, aplant cell capable of secreting such glycoprotein, and a glycoproteinhaving a human-type sugar chain secreted by this plant cell.

As a result of extensive investigations, the present inventors havefound that when a human-derived galactose transferred enzyme gene cDNAis expressed in a tobacco cultured cell BY2 strain, galactose is addedto sugar chains of most glycoproteins secreted to the exterior mediumand the glycoproteins have a sugar chain structure free of β1,2-xyloseor α1,3-fucose. The present invention has been accomplished based onthis finding. Accordingly, when a human-derived useful protein isproduced using this genetic recombinant tobacco cultured cell, theobjective protein having a sugar structure which is not an allergen, canbe secreted in the extracellular fluid.

The present invention relates to a method for the secretory productionof a glycoprotein having a human-type sugar chain. This method comprisesa step of introducing a gene of an enzyme capable of performing atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine residue and a gene of a heterologous glycoprotein,into a plant, cell to obtain a transformed plant cell, and a step ofculturing the obtained plant cell.

In the method above, the glycoprotein having a human-type sugar chainmay comprise a core sugar chain and an outer sugar chain, the core sugarchain may substantially comprise a plurality of mannoses andacetylglucosamines, and the outer sugar chain may have a terminal sugarchain moiety containing a non-reducing terminal galactose.

In the method above, the outer sugar chain may have a linear or branchedstructure.

In the method above, the branched sugar chain moiety may be a mono-,bi-, tri- or tetra-structure

In the method above, the glycoprotein may be free of fucose or xylose.

The method above for the secretory production may preferably furthercomprise a step of recovering the medium of the plant cells.

In one embodiment, the method above for the secretory production mayfurther comprise a step of adding sugar or sugar chain in vitro.

In one aspect, the present invention relates to a plant cell whichcomprises a sugar chain-adding mechanism capable of performing atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine residue and can secrete a protein with an added sugarchain added by the sugar chain-adding mechanism, wherein the sugarchain-adding mechanism adds a sugar chain comprising a core sugar chainand an outer sugar chain, the core sugar chain substantially comprises aplurality of mannoses and acetylglucosamines, and the outer sugar chainhas a terminal sugar chain moiety containing a non-reducing terminalgalactose.

The present invention further relates to a glycoprotein having ahuman-type sugar chain obtained by the method described above.

The present invention still further relates to a method for thesecretory production of a glycoprotein having a human-type sugar chain,comprising a step of introducing a gene for an enzyme capable ofperforming a transfer reaction of a galactose residue to a non-reducingterminal acetylglucosamine residue and a gene for a heterologousglycoprotein, to obtain a transformed plant cell, and a step ofexpressing the enzyme within an intracellular organelle.

The present invention still further relates to a plant cell transformedwith a gene for an enzyme capable of performing a transfer reaction of agalactose residue to a non-reducing terminal acetylglucosamine residue,wherein the enzyme is localized in the plant cell such that the plantcell is capable of synthesizing a glycoprotein having a human-type sugarchain structure.

In one aspect, in the plant cell above, the sugar chain structureincludes an added galactose residue.

In one aspect, in the plant cell above, the sugar chain structure isfree of β1,2-xylose or α1,3-fucose.

In one aspect, in the plant cell above, the sugar chain structureincludes an galactose residue added to a N-linked type sugar chain of(N-acetylglucosamine)₁₋₂(Mannose)₂₋₅(N-acetylglucosamine)₂ selected fromthe group consisting of GlcNAc₁Man₃GlcNAc₂, GlcNAc₁Man₅GlcNAc₂,GlcNAc₂Man₃GlcNAc₂ and GlcNAc₁Man₄GlcNAc₂.

In one aspect, in the plant cell above, the enzyme is localized withinan intracellular organelle in the plant cell.

The present invention still further relates to a plant regenerated fromthe plant cell above.

The present invention still further relates to a seed produced from theplant above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing a process for cloning hGT.

FIG. 2 is a schematic view showing a process for constructing vectorpGAhGT for expressing hGT.

FIG. 3 is a photograph showing the Southern analysis of genome oftransformant tobacco cultured cell. FIG. 3(A) shows the results whengenomic DNA (40 μg) was digested by EcoRI and HindIII and thenelectrophoresed. The numerals in the left side show the sites of the DNAmolecular weight marker. FIG. 3(B) shows a schematic view of fragment2.2 kb containing promoter, hGT and terminator integrated into eachtransformant.

FIG. 4A is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium by high-performance liquid chromatography.

FIG. 4B is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium by high-performance liquid chromatography.

FIG. 5 is a view showing the structures and analysis results of sugarchains in the glycoprotein secreted in the GT6 strain culture medium.The numerals in parentheses in the Figure show the molar ratio of sugarchain having each structure shown in the Figure.

FIG. 6 is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 7 is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 8 is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 9 is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 10 is a view showing the IS-MS/MS analysis of PA-sugar chainprepared from GT6 strain culture medium. B is a partial enlarged view ofA.

FIG. 11 is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 12 is a view showing the analysis of PA-sugar chain prepared fromGT6 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 13 is a photograph showing the results of sialic acid transferasereaction in vitro using GT6 stain culture medium-derived glycoprotein,BY2 strain culture medium-derived glycoprotein or asialo fetuin as thesubstrate.

FIG. 14A is a view showing the analysis of PA-sugar salt prepared fromBY2 strain culture medium by high-performance liquid chromatography.

FIG. 14B is a view showing the analysis of PA-sugar salt prepared fromBY2 strain culture medium by high-performance liquid chromatography.

FIG. 15A is a view showing the structures and analysis results of sugarchains in the glycoprotein secreted in the GT6 strain culture medium.The numerals in parentheses in the Figure show the molar ratio of sugarchain having each structure shown in the Figure.

FIG. 15B is a view showing the structures and analysis results of sugarchains in the glycoprotein secreted in the GT6 strain culture medium.The numerals in parentheses in the Figure show the molar ratio of sugarchain having each structure shown in the Figure.

FIG. 16 is a view showing the analysis of PA-sugar chain prepared fromBY2 strain culture medium.

FIG. 17 is a view showing the analysis of PA-sugar chain prepared fromBY2 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 18A is a view showing the analysis of PA-sugar chain prepared fromBY2 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 18B is a view showing the analysis of PA-sugar chain prepared fromBY2 strain culture medium and the exoglycosidase digestion productthereof by high-performance liquid chromatography.

FIG. 19 is a photograph showing the results of isoelectric focusingelectrophoresis. Proteins in the spent media of cultured tobacco cellswere analyzed by isoelectric focusing and stained for peroxidaseactivity. Wild Type denotes BY2 strain. WT-HRP denotes transformant ofBY2 strain with HRP. GT-HRP denotes the transformant of GT6 strain withHRP gene.

FIG. 20 is a photograph showing the results of lectin staining ofproteins in the spent media of transgenic cultured tobacco cells.Proteins were fractionated by SDS-PAGE and stained with Coomassiebrilliant blue (A), or transferred to nitrocellulose membrane andtreated with ConA(B), and RCA120(C). Wild Type denotes BY2 strain.WT-HRP-2 denotes one of the transformants of BY2 strain with HRP gene.GT-HRP-5 denotes one of the transformants of GT6 strain with HRP gene.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail below.

Herein, unless otherwise specified, protein separating and analyzingmethods and immunological techniques known in the art can be employed.These techniques can be performed using commercially available kits,antibodies, marker substances and the like.

The method of the present invention is a method for producing aglycoprotein having a human type sugar chain. In the present invention,the “human-type sugar chain” means a sugar chain having a galactoseresidue bonded to an N-acetylglucosamine residue. The galactose residuein the human-type sugar chain may be a terminal of the sugar chain or asialic acid residue may further be bonded to the outer side of thegalactose residue. In the glycoprotein having a human-type sugar chaincomprising a core sugar chain moiety, a branched sugar chain moiety anda terminal sugar chain moiety of the present invention, at least one ofxylose and fucose is preferably not bonded in one or more of themoieties, more preferably in any of the moieties. Most preferably, thehuman-type sugar chain contains neither xylose nor fucose.

The plant cell may be any plant cell. The plant cell may have any formof cultured cell, cultured tissue, cultured organ and plant body. Amongthese, preferred are cultured cell, cultured tissue and cultured organ,more preferred is cultured cell. The plant species which can be used inthe production method of the present invention may be any plant specieswhich can perform the genetic transduction. Examples of the plantspecies which can be used in the production method of the presentinvention include plants belonging to Solanaceae, Gramineae, Cruciferae,Rosaceae, Leguminosae, Cucurbitaceae, Labiatae, Liliaceae,Chenopodiaceae and Umbelliferae.

Examples of the Solanaceae plant include plants belonging to Nicotiana,Solanum, Datura, Lynopersion or Petunia, such as tobacco, egg-plant,potato, tomato, red pepper and petunia.

Examples of the Gramineae plant include plants belonging to Oryza,Hordenum, Secale, Sccaharum, Echinochloa or Zea, such as rice, barley,rye, barnyard millet, sorghum and corn.

Examples of the Cruciferae plant include plants belonging to Raphanus,Brassica, Arabidopsis, Wasabia or Capsella, such as radish, rape,whitlowgrass, horseradish and shepherd's purse.

Examples of the Rosaceae plant include plants belonging to Orunus,Malus, Pynus, Fragaria or Rosa, such as Japanese apricot, peach, apple,pear, strawberry and rose.

Examples of the Leguminosae plant include plants belonging to Glycine,Vigna, Phaseolus, Pisum, Vicia, Arachis, Trifolium, Alphalfa orMedicago, such as soybean, red bean, kidney bean, green pea, horsebean,peanut, clover and bur clover.

Examples of the Cucurbitaceae plant include plants belonging to Luffa,Cucurbita or Cucumis, such as luffa, cushaw, cucumber and melon.

Examples of the Labiatae plant include plants belonging to Lavandula,Mentha or Perilla, such as lavender, mint and perilla.

Examples of the Liliaceae plant include plants belonging to Allium,Liluum or Tulipa, such as Welsh onion, garlic, lily and tulip.

Examples of the Chenopodiaceae plant include plants belonging toSpinacia, such as spinach.

Examples of the Umbelliferae plant include plants belonging to Angelica,Daucus, Cryptotaenia or Apitum, such as Angelica polyclada, carrot,trefoil and celery.

Among these plants for use in the production method of the presentinvention, preferred are tobacco, tomato, potato, rice, corn, radish,soybean, green pea, bur clover and spinach, more preferred are tobacco,tomato, potato, corn and soybean.

The “enzyme capable of performing a transfer reaction of a galactoseresidue to a non-reducing terminal acetylglucosamine residue” means anenzyme which can transfer a galactose residue to a non-reducing terminalacetylglucosamine residue generated at the addition of a sugar chainafter the synthesis of the protein moiety of a glycoprotein within aplant cell. Examples of such an enzyme include galactosyl transferase,lactose synthase and β-galactosidase. Such an enzyme may be derived fromany animal species but is preferably derived from mammals, morepreferably from human.

This enzyme is preferably localized in an intracellular organelle.Although restriction to a specific theory is not intended, the presentinventors consider that this enzyme being present in an intracellularorganelle, such as endoplasmic reticulum and Golgi body, thereby actingon a protein or sugar chain before a fucose or xylose residue is added,or acting so as to inhibit the addition of a fucose or xylose residue,at the time of expression and secretion of a heterologous glycoproteinin plant cells.

The “enzyme gene capable of performing a transfer reaction of agalactose residue to a non-reducing terminal acetylglucosamine residue”may be isolated from any animal cell using a nucleotide sequence knownto code this enzyme, or a commercially available enzyme may be purchasedor may be used after modifying it to suit to the expression in plants.

Herein, the “gene” means the structural gene moiety. In order tofacilitate the expression in plants, a regulator sequence such aspromoter, operator and terminator may be linked to the gene.

The “heterologous glycoprotein” means a glycoprotein which is originallynot expressed in plants for use in the present invention. Examples ofthe heterologous glycoprotein include enzyme, hormone, cytokine,antibody, vaccine, receptor and serum protein. Examples of the enzymeinclude horseradish peroxidase, kinase, glucocerebrosidase,α-galactosidase, phytase, TPA (tissue-type plasminogen activator) andHMG-CoA reductase. Examples of the hormone and cytokine includeenkephalin, interferon alpha, GM-CSF, G-CSF, chorionic gonadotropichormone, interleukin-2, interferon beta, interferon gamma,erythropoietin, vascular endothelial growth factor, human chorionicgonadotropin (HCG), luteinizing hormone (LH), thyroid-simulating hormone(TSH), prolactin and follicle-stimulating hormone. Examples of theantibody include IgG, scFv and secretory IgA. Examples of the vaccineinclude hepatitis B surface antigen, rotavirus antigen, Escherichia colienterotoxin, malaria antigen, G protein of rabies virus, and HIV virusglycoprotein (e.g., gp120). Examples of the receptor and matrix proteininclude EGF receptor, fibronectin, α1-antitrypsin and coagulation factorVIII. Examples of the serum protein include albumin, complement systemprotein, plasminogen, corticosteroid-binding globulin, Thyroxine-bindingglobulin and protein C.

The “gene of heterologous glycoprotein” may be isolated from any cellusing a nucleotide sequence known to code the objective heterologousglycoprotein or a commercially available gene may be purchased or may beused after modifying it to suit to the expression in plants.

The gene of an enzyme capable of performing a transfer reaction of agalactose residue to a non-reducing terminal acetylglucosamine residueand the gene of heterologous glycoprotein are introduced into a plantcell by a method known in the art. These genes may be introducedseparately or simultaneously. Those skilled in the art will appreciatethat the choice of method might depend on the type of plant targeted fortransformation.

Suitable methods of transforming plant cells include microinjection(Crossway et al., BioTechniques 4:320-334 (1986)), electroporation(Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986),Agrobacterium-mediated transformation (Hinchee et al., Biotechnology6:915-921 (1988); See also, Ishida et al., Nature Biotechnology14:745-750 (June 1996) for maize transformation), direct gene transfer(Paszkowski et al., EMBO J. 3:2717-2722 (1984); Hayashimoto et al.,Plant Physiol 93:857-863 (1990)(rice)), and ballistic particleacceleration using devices available from Agracetus, Inc., Madison, Wis.and Dupont, Inc., Wilmington, Del. (see, for example, Sanford et al.,U.S. Pat. No. 4,945,050; and McCabe et al., Biotechnology 6:923-926(1988)). See also, Weissinger et al., Annual Rev. Genet. 22:421-477(1988); Sanford et al., Particulate Science and Technology 5.27-3791987)(onion); Svab et al., Proc. Natl. Acad. Sci. USA 87:8526-8530(1990) (tobacco chloroplast); Christou et al., Plant Physiol. 87:671-674(1988)(soybean); McCabe et al., Bio/Technology 6.923-926(1988)(soybean); Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305-4309(1988)(maize); Klein et al., Bio/Technology 6:559-563 (1988) (maize);Klein et al., Plant Physiol. 91:440-444 (1988) (maize); Fromm et al.,Bio/Technology 8:833-839 (1990); and Gordon-Kamm et al., Plant Cell 2:603-618 (1990) (maize); Koziel et al., Biotechnology 11: 194-200 (1993)(maize); Shimamoto et al., Nature 338: 274-277 (1989) (rice); Christouet al., Biotechnology 9: 957-962 (1991) (rice); Datta et al.,Biol/Technology 3:736-740 (1990) (rice); European Patent Application EP0 332 581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology11: 1553-1558 (1993) (wheat); Weeks et al., Plant Physiol. 102:1077-1084 (1993) (wheat); Wan et al., Plant Physiol. 104: 37-48 (1994)(barley); Jahne et al., Theor. Appl. Genet. 89:525-533 (1994) (barley);Umbeck at al., Bio/Technology 5: 263-266 (1987) (cotton); Casas et al.,Proc. Natl. Acad. Sci. USA 90:11212-11216 (December 1993) (sorghum);Somers et al., Bio/Technology 10:1589-1594 (December 1992) (oat);Torbert et al., Plant Cell Reports 14:635-640 (1995) (oat); Weeks etal., Plant Physiol. 102:1077-1084 (1993) (wheat); Chang et al., WO94/13822 (wheat) and Nehra et al., The Plant Journal 5:285-297 (1994)(wheat). A particularly preferred set of embodiments for theintroduction of recombinant DNA molecules into maize by microprojectilebombardment can be found in Koziel et al., Biotechnology 11: 194-200(1993), Hill et al., Euphytica 85:119-123 (1995) and Koziel et al.,Annals of the New York Academy of Sciences 792:164-171 (1996). Anadditional preferred embodiment is the protoplast transformation methodfor maize as disclosed in EP 0 292 435. Transformation of plants can beundertaken with a single DNA species or multiple DNA species (i.e.co-transformation) and both these techniques are suitable for use withthe peroxidase coding sequence.

The gene product expressed and secreted by the plant cell havingincorporated thereinto the above-described genes can be identified by amethod known in the art. Examples of the identification method includesilver staining, Western blotting, Northern hybridization and detectionof enzymatic activity.

The transformed cell expressing an enzyme capable of performing atransfer reaction of a galactose residue into a non-reducing terminalacetylglucosamine residue and expressing a heterologous glycoprotein,expresses and secretes a heterologous glycoprotein having a human-typesugar chain. In other words; the thus-obtained transformed plant has ahuman-type sugar chain-adding mechanism and by culturing thistransformed cell, human-type glycoprotein can be expressed and secretedin a large amount in the medium.

This human-type glycoprotein comprises a core sugar chain and an outersugar chain, and the core sugar chain substantially comprises aplurality of mannoses and acetylglucosamines. The outer sugar chain ofthe glycoprotein obtained contains a non-reducing terminal sugar chainmoiety. The outer sugar chain may have a linear structure or a branchedstructure. The branched sugar chain moiety may be any of mono-, bi-,tri- and tetra-structures. The glycoprotein produced by the transformedcell preferably is free of fucose or xylose.

The resulting transformed plant cell may be maintained in the state ofcultured cell, may be differentiated into a specific tissue or organ, ormay be regenerated in a complete plant body or in a part such as seed,fruit, leaf, root, stem or flower obtained from a complete plant body.

For the culture, differentiation or regeneration of the transformedplant cell, means and culture mediums known in the art are used.Examples of the medium include Murashige-Skoog (MS) medium, Gamborg B5(B) medium, White medium and Nitsch & Nitsch (Nitsch) medium, however,the present invention is not limited thereto. These mediums are usuallyused after adding thereto an appropriate amount of a plant growthcontrol substance (e.g., plant hormone) and the like.

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., Plant Tissue Culture Letters, 2:74,1985; Toriyama et al., Theor. Appl. Genet., 73:16, 1986; Yamada et al.,Plant Cell Rep., 4:85, 1986;. Abdullah et al., Biotechnology, 4:1087,1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, Biotechnology,6:397, 1988).

Agrobacterium-mediated transfer is also a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described above.

Fundamentally, as long as the transformed plant cell grows and thenexpresses and secretes the desired gene product, a culture medium havingany composition containing trace nutrients necessary for the growth ofplant cells, such as carbon source, nitrogen source, vitamins and salts,may be used for the secretory production of a glycoprotein having ahuman-type sugar chain. Also, polyvinylpyrrolidone, protease inhibitorand the like may be added so as to stabilize the secreted heterologousprotein and attain efficient secretion of the heterologous protein.

The glycoprotein having a human-type sugar chain expressed and secretedby the transformed plant cell can be typically isolated from the mediumof plant cells. The isolation of glycoprotein from the medium of plantcells can be performed using a method well-known to one skilled in theart. For example, the glycoprotein can be purified to isolate it fromthe medium using, individually or in combination, techniques such assalting out (e.g., ammonium sulfate precipitation, sodium phosphateprecipitation), solvent precipitation (e.g., protein fractionalprecipitation by acetone or ethanol), dialysis, gel filtration, ionexchange, column chromatography such as reverse phase, ultrafiltration,and high-performance chromatography (HPLC).

Alternatively, the glycoprotein of the present invention may also beisolated or extracted from plant cells. Furthermore, the glycoprotein ofthe present invention, which is contained in transformed cells, can beused as it is for food. The glycoprotein of the present invention has ahuman-type sugar chain addition and therefore, is free of antigenicityand suitable for the administration to animals including humans.

Further also included within the present invention, the plant cell orthe plant body transformed with a gene of an enzyme capable ofperforming a transfer reaction of a galactose residue to a non-reducingterminal acetylglucosamine residue, may have inactivated or suppressedactivity of β1,2-xylose transfer enzyme or α1,3-fucose transfer enzyme.

Strasser et al. has isolated cDNA encoding β1,2-xylosyltransferase fromArabidopsis thaliana (Strasser R, Mucha J, Mach L, Altmann F, Wilson IB, Glossl J, Steinkellner H, Molecular cloning and functional expressionof β1,2-xylosyltransferase cDNA from Arabidopsis thaliana. FEB S Lett.(2000) 472: 105-108). Homology searching against databases such as NIHGenBank may show nucleotide sequences corresponding to some EST(expression sequence tag) clones and genome sequence in some plants, aresimilar to the nucleotide sequence encoding Arabidopsisβ1,2-xylosyltransferase. Based on these sequences, nucleic acid encodingβ1,2-xylosyltransferase may be cloned from the host plant and used toestablish a plant cell or plant with reduced xylosyltransferase activityby repressing β1,2-xylosyltransferase gene expression. Repression ofβ1,2-xylosyltransferase gene expression may be carried out by anantisense method, co-suppression method, RNAi method or so on.

Alternatively, a plant cell or plant with reduced xylosyltransferaseactivity may be established by chemical mutagenesis, site-directedmutagenesis using oligonucleotides, tagging methods or so on. In saidplant, introduction and expression of one or more glycosyltransferasegenes encoding galactose transferase together with a gene encoding aheterologous polypeptide may produce a heterologous polypeptide withhumanized glycan structure or excrete a heterologous polypeptide withhumanized glycan structure.

Similarly, Wilson et al. isolated cDNA encoding α1,3-fucosyltransferasefrom Arabidopsis thaliana (Wilson I B, Rendic D, Freilinger A, Dumic J,Altmann F, Mucha J, Muller S, Hauser M T Cloning and expression of cDNAsencoding α1,3-fucosyltransferase homologues from Arabidopsis thaliana.Biochim Biophys Acta (2001) 1527:88-96). Leiter et al. isolated cDNAencoding α1,3-fucosyltransferase from mung bean (Leiter, H., Mucha, J.,Staudacher, E., Grimm, R., Glossl, J., Altmann, F. Purification, cDNAcloning, and expression of GDP-L-Fuc:Asn-linked GlcNAcα1,3-fucosyltransferase from mung beans. J. Biol. Chem. (1999)274:21830-21839). Homology searching against databases such as NIHGenBank may show nucleotide sequences corresponding to some EST(expression sequence tag) clones and genome sequence in some plants, aresimilar to the nucleotide sequence encoding Arabidopsisα1,3-fucosyltransferase. Based on these sequences, nucleic acid encodingα1,3-fucosyltransferase may be cloned from the host plant and used toestablish a plant cell or plant with reduced fucosyltransferase activityby repressing α1,3-fucosyltransferase gene expression. Repression ofα1,3-fucosyltransferase gene expression can be carried out by anantisense method, co-suppression method, RNAi method or so on.

Alternatively, a plant cell or plant with reduced fucosyltransferaseactivity may be established by chemical mutagenesis, site-directedmutagenesis using oligonucleotides, tagging methods or so on. In saidplant, introduction and expression of one or more glycosyltransferasegenes encoding galactose transferase together with a gene encoding aheterologous polypeptide may produce a heterologous polypeptide withhumanized glycan structure or excrete a heterologous polypeptide withhumanized glycan structure.

EXAMPLES

The present invention is described below by referring to the Examples.The following Examples are only to illustrate but not to limit thepresent invention.

1. Cloning of Human β1-4 Galactose Transferase Gene

The β1-4 galactose transferase (hGt) (EC2.4.1.38) has been alreadycloned and a primary structure comprising 400 amino acids has beenrevealed (K. A. Masri et al., Biochem. Biophys. Res. Commun., 157,657-663 (1988)).

(1) Primer Preparation and Template DNA

By referring to the report of Masri et al., the following primer wereprepared.

hGT-5Eco: (SEQ. ID NO: 1) 5′-AAAGAATTCGCGATGCCAGGCGCGCGTCCCT-3′hGT-2Sal: (SEQ. ID. NO: 2) 3′-TCGATCGCAAAACCATGTGCAGCTGATG-5′ hGT-7Spe:(SEQ. ID. NO: 3) 3′-ACGGGACTCCTCAGGGGCGATGATCATAA-5′ hGT6Spe:(SEQ. ID. NO: 4 5′-AAGACTAGTGGGCCCCATGCTGATTGA-3′

As the template DNA, human genomic DNA, human placenta cDNA and humankidney cDNA purchased from Clontech were used.

(2) Cloning of hGT Gene cDNA

Using two combinations of (i) template of human genomic DNA with primersof hGT-5Eco and hGT-7Spe and (ii) template of human placenta cDNA withprimers of hGT-2Sal and hGT6Spe, a PCR reaction was performed under thefollowing conditions to obtain fragments of 0.4 kb and 0.8 kb containingan hGT-coding region.

(PCR Reaction System)

Water was added to 1 μl of template DNA, 5 μl of 10×PCR buffer, 4 μl ofdNTPs (200 μM), primer (10 pmol) and 0.5 μl of Tag polymerase (producedby Takara Shuzo) (in the case of Tub polymerase, 0.2 μl) to make 50 μl.

(PCR Reaction Condition)

First Stage:

cycle number: 1, denaturation (94° C.): 5 min., annealing (55° C.): 1min., extension (72° C.): 2 min.

Second Stage:

cycle number: 30, denaturation (94° C.): 1 min., annealing (55° C.): 1min., extension (72° C.): 2 min.

Third Stage:

cycle number: 1, denaturation (94° C.): 1 min., annealing (55° C.): 2min., extension (72° C.): 5 min.

Two fragments obtained were combined to construct hGT gene cDNA andsubcloned into pBluescriptIISK+(SK). The pBluescriptIISK+(SK) waspurchased from Stratagene. FIG. 1 shows the construction of plasmidcontaining hGT gene cDNA. SEQ. ID. NO:5 shows the base sequence of theobtained hGT gene and SEQ. ID. NO:6 shows the presumed amino acidsequence.

The obtained sequence was different from the hGT sequence disclosed inMasri et al. supra. in the following points: a) A at the position 528, Cat the position 562 and A at the position 1047 were changed to G, T andG, respectively, but the amino acids coded were not changed; b) 9 basesat the positions 622 to 630 were deleted; and c) in order to connect theabove-described fractions of 0.4 kb and 0.8 kb, G at the position 405and T at the position 408 were transformed to A and A, respectively, atthe preparation of primer.

Incidentally, the hGT gene cDNA which has two initiation codons (ATG),was designed in this experimentation such that the translation startsfrom the second initiation codon (position 37).

2. Introduction of hGT Gene into Tobacco Cultured Cell

(1) hGT has been reported to be Escherichia coil expressed as an activetype (see, D. Aoki et al., EMBO J., 9, 3171 (1990) and K. Nakazawa etal., J. Biochem., 113, 747 (1993)).

In order to express hGT in tobacco cultured cells, a vector pGAhGT forexpression was constructed as shown in FIG. 2. The promoter used wascauliflower mosaic virus 35S promoter (CaMV 35S promoter) and theselectable marker used was kanamycin resistant gene. The pGAhGT wasintroduced into tobacco cultured cells through Agrobacterium.

The transformation of Agrobacterium was performed using a triparentalmating method by Bevan et al (see, M. Bevan, Nucleic Acid Res., 12, 8711(1984)). Escherichia coli DH 5α strain (suE44, ΔlacU169 (φ801acZΔM15),hsdR17) (Bethesda Research Laboratories Inc., Focus 8(2), 9(1986))having pGA-type plasmid (see, G. An, Methods Enzymol. 153, 292 (1987))and Escherichia coli HB101 having helper plasmid pRK2013 (see, M. Bevan,Nucleic Acid Res., 12, 8711 (1984)) each was cultured at 37° C.overnight in 2×YT medium containing 12.5 mg/L of tetracycline and 50mg/L of kanamycin, and Agrobacterium tumefaciens EHA101 strain (see, E.H. Elizabeth, J. Bacteriol., 168, 1291 (1986)) was cultured at 28° C.over two nights in 2×YT medium containing 50 mg/L of kanamycin and 25mg/L of chloramphenicol. From each culture medium, 1.5 ml wastransferred into an Eppendorf tube and cells were collected and washedthree times with LB medium. The obtained cells of each species weresuspended in 100 μl of 2×YT medium, these three kinds of cells weremixed, the mixture was smeared on 2×YT agar medium and cultured at 28°C. to conjugation-transmit the pGA-type plasmid from Escherichia coli toAgrobacterium. After 2 days, a part of cells grown throughout thesurface of the 2×YT agar medium were scraped by a loop and coated on LBagar medium containing 50 mg/L of kanamycin, 12.5 mg/L of tetracyclineand 25 mg/L of chloramphenicol. After culturing at 28° C. for 2 days, asingle colony was selected.

The transformation of tobacco cultured cells was performed by the methoddescribed in G. An, Plant Mol. Bio. Manual, A3, 1. A suspension ofAgrobacterium (EHA101 strain having pGA-type plasmid) cultured at 28° C.for 36 hours in LB medium containing 12.5 mg/L of tetracycline and asuspension of tobacco cultured cells (Nicotiana tabacum L. cv. brightyellow 2) after culturing for 4 days (purchased under Cell Name of BY-2in catalogue No. RPC1 from The Institute of Physical and ChemicalResearch, Riken Gene Bank, Plant Cell Bank) were placed in a petri dishin amounts of 100 μl and 4 ml, respectively, then thoroughly mixed andleft standing at 25° C. in a dark place. After 2 days, the culturemedium in the petri dish was transferred into a centrifugation tube andthe supernatant was removed by the centrifugal separation (1,000 rpm, 5minutes). Subsequently, a fresh medium was added and after thecentrifugal separation, the cells were smeared on a plate of modified LSagar medium containing from 150 to 200 mg/L of kanamycin and 250 mg/L ofcarbenicillin and left standing at 25° C. in the dark. After about 2 to3 weeks, the callused cells were implanted on a new plate and thegrowing clones were selected. After further 2 to 3 weeks, the cloneswere transferred to 30 ml of modified LS medium having added theretokanamycin and carbenicillin and subjected to passage culture. For about1 month, the selection was repeated. From some resistant strainsobtained, 6 resistant strains (GT1, 4, 5, 6, 8 and 9) were randomlyselected.

(2) Identification of Introduced hGT Gene

The resistant strains obtained were analyzed by the Southern analysisand it was confirmed that the fragment of 2.2 kb containing CaMV35Spromoter-hGT gene cDNA-NOS terminator in T-DNA was integrated intogenomic DNA of tobacco cultured cell. From each resistant strainobtained above, genomic DNA was prepared, digested using EcoRI, HindIII,and analyzed by Southern analysis.

The preparation of chromosome DNA from tobacco cultured cells wasperformed according to the Watabe method (see, K. Watabe, “Cloning toSequence (Cloning and Sequence)”, Shokubutsu Biotechnology Jikken Manual(Plant Biotechnology Experimentation Manual), Noson Bunka Sha). 10 ml ofTobacco cultured cells were frozen by liquid nitrogen and ground intothe powder form using mortar and pestle. Before liquefaction started,about 5 g of the thus-obtained powder was added to 5 ml of 2×CTAB(cetyltrimethylammonium bromide) solution preheated to 60° C. in acentrifugal tube (40 ml) and gradually well-mixed and while occasionallymixing at 60° C. for 10 minutes or more, the temperature was maintained.Thereto, 5 ml of chloroform: isoamyl alcohol (24:1) was added andthoroughly mixed until an emulsion was formed, and the emulsion was thencentrifuged (2,800 rpm, 15 minutes, room temperature). The upper layerwas transferred to a new 40 ml-volume centrifugal tube and theextraction operation using chloroform:isoamyl alcohol (24:1) wasrepeated. To the obtained upper layer, 1/10 volume of 10% CTAB was addedand thoroughly mixed and centrifuged (2,800 rpm, 15 minutes, roomtemperature). The upper layer was transferred to a new centrifugal tubeand 1 volume of cold isopropanol was added thereto, well mixed andcentrifuged (4,500 rpm, 20 minutes, room temperature). After removingthe supernatant by an aspirator, a TE buffer solution containing 5 ml of1M sodium chloride was added and completely dissolved at 55 to 60° C.Thereto, 5 ml of cold isopropanol was added and when DNA was observed,the DNA was taken up using the end of a stick, transferred to anEppendorf tube (containing 80% cold ethanol) and rinsed. The DNA wasfurther rinsed with 70% ethanol and the dry precipitate was dissolved inan appropriate amount of TE buffer. Thereto, 5 μl of RNAaseA (10 mg/ml)was added and reacted at 37° C. for 1 hour. The 2×CTAB solution had acomposition of 2% CTAB, 0.1M Tris-HCl (pH: 8.0), 1.4M sodium chlorideand 1% polyvinyl pyrrolidone (PVP), and the 10% CTAB solution had acomposition of 10% CTAB and 0.7M sodium chloride.

The Southern analysis was performed as follows.

(i) Electrophoresis and Alkali Modification of DNA:

After completely degrading 40 μg of the obtained chromosome DNA using arestriction enzyme, 1.5% agarose gel electrophoresis (50 V) wasperformed by a standard method. The gel was stained with ethidiumbromide, photographed and shaken for 20 minutes in 400 ml of 0.25M HCl.Thereafter, the solution was discarded and the gel was immersed in 400ml of modified solution (1.5M, NaCl, 0.5M, NaOH) and gradually shakenfor 45 minutes. Subsequently, the solution was discarded and 400 ml of aneutralization solution (1.5M NaCl, 0.5M Tris-Cl (pH: 7.4)) was addedand gradually shaken for 15 minutes. After discarding the solution, 400ml of the neutralization solution was again added and gradually shakenfor 15 minutes.

(ii) Transfer:

The DNA after the electrophoresis was transferred to a nylon membrane(Hybond-N Amersham) using 20×SSC. The transfer was performed for 12hours or more. The blotted membrane was dried at room temperature for 1hour and subjected to UV fixing for 5 minutes. The 20×SSC had acomposition of 3M NaCl and 0.3M sodium citrate.

(iii) Preparation of DNA Probe

The DNA probe was prepared using Random prime Labeling Kit (produced byTakara Shuzo). In an Eppendorf tube, a reaction solution shown below wasprepared and after heating at 95° C. for 3 minutes, rapidly cooled inice: template DNA 25 ng, Random Primer 2 μl, water added to make 5 μl.10× Buffer and dNTP each in 1.5 μl and [α-³²P]dCTP (1.85 MBq, 50 mCi) in5 μl were added, followed by filling up to 24 μl with H₂O. Thereto, 1 μlof Klenow fragment was added and after keeping at 37° C. for 10 minutes,eluted through NAP10 column (produced by Pharmacia) to purify the DNA.This purified DNA was heated at 95° C. for 3 minutes and then rapidlycooled in ice to obtain a hybridization probe.

(iv) Hybridization:

To the following prehybridization solution, 0.05 mg/ml of 0.5% (w/v) SDSwas added. In the resulting solution, the membrane of (ii) above wasimmersed and the prehybridization was performed at 42° C. for 2 hours ormore. Thereafter, the DNA probe prepared in (iii) was added and thehybridization was performed at 42° C. for 12 hours or more. Theprehybridization solution had a composition of 5×SSC. 50 mM sodiumphosphate, 50% (w/v) formamide, 5×Denhardt's solution (obtained bydiluting 100×Denhardt's solution), 0.1% (w/v) SDS. The 100×Denhardt'ssolution had a composition of 2% (w/v) BSA, 2% (w/v) Ficol 400, 2% (w/v)polyvinyl pyrrolidone (PVP).

(v) Autoradiography:

After the cleaning in the following sequence, the autoradiography wasperformed by a standard method. Twice in 2×SSC and 0.1% SDS at 65° C.for 15 minutes and then, once in 0.1×SSC and 0.1% SDS at 65° C. for 15minutes.

FIG. 3 shows the results of the Southern analysis of genomic DNAprepared each resistant strain obtained above. As seen from FIG. 3, itwas confirmed that the hGT gene was integrated in four strains of GT1,6, 8 and 9.

3. Analysis of Glycoprotein Secreted by Galactosyl TransferaseTransformant

(Preparation of Glycoprotein by Extracellular Secretion of TobaccoCultured Cell GT6 Strain)

A culture medium of tobacco cultured cell GT6 strain resulting fromculturing in modified Murashige-Skoog medium prepared using mixed saltsfor Murashige-Skoog medium (produced by Wako Junyaku) for 7 days wascentrifuged at 2,000 rpm for 10 minutes at room temperature and theobtained supernatant was recovered as the GT6 strain culture medium andused in this Example. The obtained supernatant was dialyzed against dH₂O(deionized water) (1×10⁵ times dilution) and then freeze-dried.

(Preparation of N-Linked Type Sugar Chain)

The sample obtained by the freeze-drying was hydrazinolyzed at 100° C.for 10 hours to excise the sugar chain. To the hydrazinolysis product,excess acetone was added and by centrifugation at 4° C. and 10,000 rpmfor 20 minutes, sugar chains were precipitated. The sugar chains wereN-acetylated in the presence of an aqueous saturated sodiumhydrogencarbonate solution and acetic anhydride, then desalted using.Dowex 50×2 (produced by Muromachi Kagaku Kogyo), and passed throughSephadex G-25 superfine gel filter column (1.8×180 cm) equilibrated with0.1N aqueous ammonia, thereby recovering the N-linked sugar chains.

(Preparation of Pyridylaminated (PA) Sugar Chain)

The recovered N-linked sugar chains were PA-formed. The PA-sample waspassed through Sephadex G-25 super fine gel filter column (1.8×180 cm)equilibrated with an aqueous 3% acetic acid solution to purify thePA-sugar chains.

(Fractionation and Analysis of PA-Sugar Chains by HPLC)

The PA-sugar chain structure was analyzed by reversed-phase (RP) andsize-fractionation (SF) HPLC, two-dimensional sugar chain mapping usingthe exoglycosidase digestion, and IS-MS/MS analysis. In the HPLC(high-performance liquid chromatography) analysis. Jasco 880-PU HPLChaving Jasco 821-FP Intelligent Spectrofluorometer was used and thefluorescence intensity was measured at excitation wavelength of 310 nmand fluorescence wavelength of 380 nm.

In the RP-HPLC analysis using Cosmosil 5C18-P column (6×250 mm, producedby Nakaraitesc), the concentration of acetonitrile in an aqueous 0.02%TFA solution was increased from 0% to 6% over 40 minutes at a flow rateof 1.2 ml/min and thereby PA-sugar chains were eluted. In the SF-HPLCanalysis using Asahipak NH2P-50 column (4.6×250 mm, produced by ShowaDenko K.K.), the concentration of acetonitrile in a dH₂O-acetonitrilemixed solution was increased from 26% to 50% over 25 minutes at a flowrate of 0.7 ml/min and thereby PA-sugar chains were eluted.

(Analysis of PA-Sugar Chains by Exoglycosidase Digestion)

In the enzymatic digestion reaction using β-galactosidase (Diplococcuspneumoniae, Roche), each PA-sugar chain was reacted at 37° C. for 2 daysin a 0.1M sodium acetate buffer (pH: 5.5) containing 5 mU ofβ-galactosidase. Similarly, in the enzymatic digestion reaction usingN-acetylglucosamidase (Diplococcus pneumoniae, Roche), each PA-sugarchain was reacted at 37° C. for 2 days in a 0.1M sodium acetate buffer(pH: 5.5) containing 5 mU of N-acetylglucosamidase. Furthermore, in theenzymatic digestion reaction using α-mannosidase (Jack bean, Sigma),each PA-sugar chain was reacted at 37° C. for 2 days in a 0.1M sodiumacetate buffer (pH: 3.88) containing 10 mM zinc acetate and 10 μU ofα-mannosidase. Each enzymatic digestion reaction was stopped by boilingthe solution at 100° C. for 3 minutes. Then, the reaction solution wascentrifuged at 12,000 rpm for 10 minutes and the supernatant wassubjected to HPLC. The elution time of each sample sugar chain wascompared with the elution time of a known sugar chain.

(IS-MS/MS Analysis)

The IS-MS/MS Analysis was performed using Perkin-Elmer Sciex API-IIItriple-quadrupole mass spectrometer. The scan interval was 0.5 Da.

(Sialic Acid Transferase Reaction In Vitro)

The GT6 strain cell culture medium-derived glycoprotein prepared aboveafter the dialysis and freeze-drying was used as the substrate. A sialicacid transferase reaction was performed at 37° C. for 5 hours in 62.5 mMsodium cacodylate buffer solution (pH: 6.0) containing 1 mg/ml of BSA,0.5% of Triton CF-54, 2 μM of CMP-sialic acid, 6 mU of α2,6-sialyltransferase (derived from rat liver) (produced by Wako Junyaku) and 400μg of GT6 strain cell culture medium-derived glycoprotein. In thecontrol test, 400 μg of BY2 strain cell culture medium-derivedglycoprotein and 40 μg of asialo fetuin fetal (fetal bovine serum,Sigma) were used as the substrate.

(Lectin Staining)

The sialic acid transferase reaction product was subjected to SDS-PAGEat 130 V for 2 hours using 12.5% polyacrylamide gel and then transferredto nitrocellulose membrane at a constant current of 1 mA/cm² for 50minutes. In the lectin blotting, horseradish peroxidase-linked SNAlectin (produced by EY Laboratories, Inc.) 200-fold diluted with a PBSsolution containing 0.05% of Tween-20 was used. After the blotting, thestaining was performed using POD Immunostain Kit (produced by WakoJunyaku).

(Purification of Tobacco Cultured Cell GT6 Strain Culture Medium-DerivedPA-Sugar Chain)

The PA-sugar chain prepared from the GT6 strain culture medium waspurified using RP-HPLC and SF-HPLC (shown in FIG. 4A and FIG. 4B,respectively). FIG. 4A shows the peak of PA-product by RP-HPLC. Afterthe recovery of peaks (1 to 6), each was subjected to SF-HPLC (see, FIG.4B).

Exclusive of 6 peaks (shown by A to F in FIG. 4B) obtained by theSF-HPLC, the peaks were not an N-linked sugar chain. This was verifiedbecause, in IS-MS/MS analysis, signals agreeing with m/z 299.33(GlcNAc-PA) and m/z 502.52 (GlcNAc₂-PA) were not obtained. FIG. 5 showsN-linked sugar chain structures analyzed on the peaks (A-F). In FIG. 5,the numerals in parentheses indicate the molar ratio of sugar chainhaving each structure shown in the Figure.

As shown in FIG. 5, A to F are all a human-type sugar chain having agalactose residue bound to an N-acetylglucosamine residue, contain nofucose residue and except for B and D, have no xylose residue.

(Structural Analysis of Tobacco Cultured Cell GT6 Culture Medium-DerivedPA-Sugar Chain)

The molecular weight (m/z 1354.8) obtained by the IS-MS analysis of thepeak A (I in FIG. 6) agreed with GalGlcNAcMan₃GlcNAc₂-PA (1354.27). Thesignals obtained by the IS-MS/MS analysis, namely, m/z 1192.5, m/z990.5, m/z 827.5, m/z 665.5, m/z 503.0, m/z 300.0, are presumed to beGlcNAcMan₃GlcNAc₂-PA (1192.13) Man₃GlcNAc₂-PA (988.94), Man₂GlcNAc₂-PA(826.80), ManGlcNAc₂-PA (664.66), GlcNAc₂-PA (502.52) and GlcNAc-PA(299.33), respectively, and this suggests that the peak A contains thesestructures (the data are not shown).

The β-galactosidase digestion product from the peak A wasGlcNAcMan₃GlcNAc₂-PA (II in FIG. 6) and the N-acetylglucosaminidasedigestion product thereof was Man₃GlcNAc₂-PA (III in FIG. 6).

The molecular weight (m/z 1486.8) obtained by the IS-MS analysis of thepeak B (I in FIG. 7) agreed with GalGlcNAcMan₃XylGlcNAc₂-PA (1486.38).The signals obtained by the IS-MS/MS analysis, namely, m/z 1354.5, m/z1324.0, m/z 1324.0, m/z 1122.0, m/z 991.5. m/z 960.0, m/z 666.0, m/z503.0 and m/z 300.0, are presumed to be GalGlcNAcMan₃GlcNAc₂-PA(1354.27), GlcNAcMan₃XylGlcNAc₂-PA (1324.24), Man₃XylGlcNAc₂-PA(1121.05), Man₃GlcNAc₂-PA (988.94), Man₂XylGlcNAc₂-PA (958.91),ManGlcNAc₂-PA (664.66), GlcNAC₂-PA (502.52) and GlcNAc₂-PA (299.33),respectively, and this suggests that the peak B contains thesestructures (the data are not shown).

The β-galactosidase digestion product from the peak B wasGlcNAcMan₃XylGlcNAc₂-PA (II in FIG. 7) and the N-acetylglucosaminidasedigestion product thereof was Man₃XylGlcNAc₂-PA (III in FIG. 7).

The molecular weight (m/z 1355.0) obtained by the IS-MS analysis of thepeak C (I in FIG. 8) agreed with GalGlcNAcMan₃GlcNAc₂-PA (m/z 1354.27).The signals obtained by the IS-MS/MS analysis, namely, m/z 1193.5, m/z989.0. m/z 827.0, m/z 665.5, m/z 503.0, m/z 300.0, are presumed to beGlcNAcMan₃GlcNAc₂-PA (m/z 1192.13), Man₃GlcNAc₂-PA (m/z 988.94),Man₂GlcNAc₂-PA (m/z 826.80). ManGlcNAc₂-PA (m/z 664.66), GlcNAc₂-PA (m/z502.52) and GlcNAc-PA (m/z 299.33), respectively, and this suggests thatthe peak C contains these structures (the data are not shown).

The β-galactosidase digestion product from the peak C wasGlcNAcMan₃GlcNAc₂-PA (II in FIG. 8) and the N-acetylglucosaminidasedigestion product thereof was Man₃GlcNAc₂-PA (III in FIG. 8).

The molecular weight (m/z 1487.0, A in FIG. 10) obtained by the IS-MSanalysis of the peak D (I in FIG. 9) agreed withGalGlcNAcMan₃XylGlcNAc₂-PA (m/z 1486.38). The signals obtained by theIS-MS/MS analysis, namely, m/z 1354.0, m/z 1325.0, m/z 1191.0, m/z1121.5, m/z 989.5, m/z 828.5, m/z 503.0 and m/z 300.5, are presumed tobe GalGlcNAcMan₃GlcNAc₂-PA (m/z 1354.27), GlcNAcMan₃XylGlcNAc₂-PA (m/z1324.24), GlcNAcMan₃GlcNAc₂-PA (m/z 1192.13), Man₃XylGlcNAc₂-PA (m/z1121.05), Man₃GlcNAc₂-PA (m/z 988.94), Man₂GlcNAc₂-PA (m/z 826.80),GlcNAc₂-PA (m/z 502.52) and GlcNAc-PA (m/z 299.33), respectively, andthis suggests that the peak D contains these structures (B in FIG. 10).

The β-galactosidase digestion product from the peak D wasGlcNAcMan₃XylGlcNAc₂-PA (II in FIG. 9) and the N-acetylglucosaminidasedigestion product thereof was Man₃XylGlcNAc₂-PA (III in FIG. 9).

The molecular weight (m/z 1516.6) obtained by the IS-MS analysis of thepeak E (I in FIG. 11) agreed with GalGlcNAcMan₄GlcNAc₂-PA (1516.41). Thesignals obtained by the IS-MS/MS analysis, namely, m/z 1355.0, m/z1193.0, m/z 990.0, m/z 826.5, m/z 665.0, m/z 503.5 and m/z 300.0, arepresumed to be GalGlcNAcMan₃GlcNAc₂-PA (m/z 1354.27),GalNAcMan₃GlcNAc₂-PA (m/z 1192.13), Man₃GlcNAc₂-PA (m/z 988.94),Man₂GlcNAc₂-PA (m/z 826.80), ManGlcNAc₂-PA (m/z 664.66) GlcNAc₂-PA (m/z502.52) and GlcNAc-PA (m/z 299.33), respectively, and this suggests thatthe peak E contains these structures (the data are not shown).

The β-galactosidase digestion product from the peak E wasGlcNAcMan₄GlcNAc₂-PA (II in FIG. 11) and the N-acetylglucosaminidasedigestion product thereof was Man₄GlcNAc₂-PA (III in FIG. 11).

The molecular weight (m/z 1679.8) obtained by the IS-MS analysis of thepeak F (I in FIG. 12) agreed with GalGlcNAcMan₅GlcNAc₂-PA (1678.55). Thesignals obtained by the IS-MS/MS analysis, namely, m/z 1517.5, m/z1313.5, m/z 1152.0, m/z 827.5, m/z 665.5, m/z 503.0 and m/z 300.0, arepresumed to be GlcNAcMan₃GlcNAc₂-PA (m/z 1516.41). Man₃GlcNAc₂-PA(1313.22), Man₄GlcNAc₂-PA (1151.08), Man₂GlcNAc₂-PA (826.80),ManGlcNAc₂-PA (664.66), GlcNAc₂-PA (502.52) and GlcNAc-PA (m/z 299.33),respectively, and this suggests that the peak F contains thesestructures (the data are not shown).

The β-galactosidase digestion product from the peak F wasGlcNAcMan₅GlcNAc₂-PA (II in FIG. 12) and the N-acetylglucosaminidasedigestion product thereof was Man₅GlcNAc₂-PA (III in FIG. 12).

From these results, the peak A or C is considered to be eitherα-D-Man-(1→6) [β-D-Gal-(1→4)-β-D-GlcNAc-(1→2)-α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GalGN₁M3-PA) or[β-D-Gal-(1→4)-β-D-GlcNAc(1→2)-α-D-Man-(1→6)] α-D-Man-(1→3)β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GalGN¹M3-PA).

The peak B or D is considered to be either α-D-Man-(1→6)[β-D-Gal-(1→4)-β-D-GlcNAc-(1→2)-α-D-Man-(1→3)] [β-D-Xyl-(1→2)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GalGN₁M3X-PA) or[β-D-Gal-(1→4)-β-D-GlcNAc(1→2)-α-D-Man-(1→6)] [α-D-Man-(1→3)][β-D-Xyl-(1→2)] β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAC-PA (GalGN¹M3X-PA).

The peak E is considered to be either α-D-Man-(1→6)-α-D-Man-(1→6)[β-D-Gal-(1→4)-β-D-GlcNAc-(1→2)-α-D-Man-(1→3)] β-D-Man-(1→4)β-D-GlcNAc-(1→4)-GlcNAc-PA (GalGNM4-PA) or α-D-Man-(1→3)-α-D-Man-(1→6)[β-D-Gal-(1→4)-β-D-GlcNAc(1→2)-α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GalGNM4-PA).

The peak F is considered to be α-D-Man-(1→6) [α-D-Man-(1→3)]α-D-Man-(1→6) [β-D-Gal-(1→4)-β-D-GlcNAc(1→2)-α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GalGNM5-PA).

(Sialic Acid Transferase Reaction In Vitro)

A sialic acid transferase reaction in vitro was performed using GT6strain culture medium-derived glycoprotein, BY2 strain culturemedium-derived glycoprotein or asialo fetuin as the substrate. Eachreactant was transferred to nitrocellulose membrane and subjected tolectin staining. As a result, the lectin staining was positive in thecase where the substrate was asialo fetuin (lane 1 in FIG. 13) or GT6strain culture medium-derived glycoprotein (lane 2 in FIG. 13), and thelectin staining was negative in the case where the substrate was BY2strain culture medium-derived glycoprotein (lane 3, FIG. 13). From this,it is revealed that tobacco cultured cell GT6 strain culturemedium-derived glycoprotein acts as the substrate in the sialic acidtransferase reaction.

COMPARATIVE EXAMPLE Analysis of Glycoprotein Secreted byNon-Transformant BY2 Strain Cultured Cell

(Preparation of Glycoprotein by Extracellular Secretion)

A freeze-dried culture supernatant sample was obtained by the samemethod as in GT6 strain except for using BY2 strain in place of GT6strain.

(Preparation of N-Linked Sugar Chain)

N-Linked sugar chains were recovered by the same method as in GT6 strainexcept for using TSK gel TOYO PERAL HW-40 (produced by TOSO) gel filtercolumn (2.5×30 cm) in place of the Sephadex G-25 super fine gel filercolumn (1.8×180 cm).

(Preparation of Pyridylaminated (PA) Sugar Chain)

The recovered N-linked sugar chains were PA-formed using2-aminopyridine. The PA-sample was filtered through TSK gel TOYO PERALHW-40 (produced by TOSO) gel filter column (2.5×30 cm) to purify thePA-sugar chains.

(Fractionation and Analysis of PA-Sugar Chain by HPLC)

The fractionation and analysis of PA-sugar chains were performed in thesame manner as in GT6 strain except for using HITACHI HPLC system havinga HITACHI FL Detector L-7480 in place of Jasco 880-PU HPLC having aJasco 821-FP Intelligent Spectrofluorometer.

(Analysis of PA-Sugar Chain by Exoglycosidase Digestion)

In the enzymatic digestion reaction using N-acetylglucosaminidase(Diplococcus pneumoniae, Roche), each PA-sugar chain was reacted at 37°C. for 2 days in a 0.1M sodium acetate buffer (pH: 5.45) containing 3 mUof N-acetylglucosaminidase. In the enzymatic digestion reaction usingα-mannosidase (Jack bean, Sigma), each PA-sugar chain was reacted at 37°C. for 2 days in a 0.1M sodium acetate buffer (pH: 4.0) containing 10 mMzinc acetate and 10 μU of α-mannosidase. Each enzymatic digestionreaction was stopped by boiling the solution at 100° C. for 3 minutes.Then, the reaction solution was centrifuged at 12,000 rpm for 10 minutesand the supernatant was subjected to HPLC. The elution time of eachsample sugar chain was compared with the elution time of a known sugarchain.

(IS-MS/MS Analysis)

This analysis was performed in the same manner as in GT6 strain.

(Preparation of BY2 Strain Culture Medium-Derived PA-Sugar Chain)

The PA-sugar chains prepared from BY2 culture medium were also purifiedusing RP-HPLC and SF-HPLC (FIGS. 14A and 14B). FIG. 14A shows the peaksof PA-product by RP-HPLC. After the recovery, each fraction (I to X) wassubjected to SF-HPLC (FIG. 14B).

In the fractions (I, II and III) shown in FIG. 14A, N-linked sugar chainwas not detected by SF-HPLC analysis. This was verified because inIS-MS/MS analysis, signals agreeing with m/z 299.33 (GlcNAc-PA) and m/z502.52 (GlcNAc₂-PA) were not obtained. The peaks of fractions IV to Xwere analyzed by SF-HPLC, as a result, 21 peaks were obtained (see, FIG.14B). FIGS. 15A and 15B show analyzed N-linked sugar chain structures.

(Structural Analysis of BY2 Strain Culture Medium-Derived PA-SugarChain)

As a result of IS-MS/MS analysis of the peaks V-4, VII-3 and VIII-4shown in FIG. 14B, the molecular weights (m/z) were 1639.0, 1476.5 and1314.5, respectively and, on considering the elution sites in SF-HPLCtogether, the sugar chain structures present in the peaks agreed withPA-high mannose sugar chain Man₇GlcNAc₂-PA, Man₆GlcNAc₂-PA andMan₅GlcNAc₂-PA, respectively (the data are not shown).

Also, as a result of SF-HPLC analysis, the α-mannosidase digestionproduct of sugar chain of each peak agreed with Man GlcNAc₂-PA (M1) (thedata are not shown). When the isomers M7A, M7B and M7D of Man₇GlcNAc₂-PAwere compared, the elution site of the peak V-4 agreed with the elutionsite of M7A. Among the isomers M6B and M6C of Man₆GlcNAc₂-PA, the peakVII-3 agreed with M6B. Also, the isomer M5A of Man₅GlcNAc₂-PA and thepeak VIII-4 agreed.

From these data, the peaks V-4, VII-3 and VIII-4 shown in FIG. 14B were,as shown in FIG. 15A, α-D-Man-(1→2)-α-D-Man-(1→6) [α-D-Man-(1→3)]α-D-Man-(1→6) [α-D-Man-(1→2)-α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (M7A), α-D-Man-(1→6)[α-D-Man-(1→3)] α-D-Man-(1→6) [α-D-Man(1→2)-α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (M6A), and α-D-Man-(1→6)[α-D-Man-(1→3)] α-D-Man-(1→6) [α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (M5A). In the figure, thenumerals in parentheses indicate the molar ratio of sugar chain havingeach structure shown in the Figure.

The molecular weight (m/z 1267.5) determined by the IS-MS analysis ofthe peaks IV-1 and V-1 shown in FIG. 14B agreed with the calculatedvalue of Man₃XylFucGlcNAc₂-PA (M3FX: 1267.19). Also, the elution site inHPLC completely agreed with M3FX standard product on the two-dimensionalsugar chain map. Furthermore, as a result of SF-HPLC analysis andIS-MS/MS analysis, the α-mannosidase digestion product of sugar chain ofeach peak agreed with the calculated value of ManXylFucGlcNAc₂-PA (MFX:942.91) (see, FIGS. 17A and 17B).

From these data, the peaks IV-1 and V-1 shown in FIG. 14B were, as shownin FIG. 15B, α-D-Man-(1→6) [α-D-Man-(1→3)] [β-D-Xyl-(1→2)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-[α-L-Fuc-(1→3)]GlcNAc-PA (M3FX).

The molecular weight (m/z 1417.0) determined by the IS-MS analysis ofthe peak VII-2 shown in FIG. 14B agreed with the calculated value ofGlcNAcMan₃XylFucGlcNAc₂-PA (GNM3FX: 1470.38). Also, as a result ofSF-HPLC analysis and IS-MS/MS analysis, the N-acetylglucosaminidasedigestion product of sugar chain of the peak agreed withMan₃XylFucGlcNAc₂-PA (M3FX: 1267.19). Furthermore, after the digestionby α-mannosidase, the SF-HPLC analysis and IC-MS/MS analysis revealedthat the product agreed with ManXylFucGlcNAc₂-PA (MFX: 942.91) (C to Ein FIG. 17). On the two-dimensional sugar chain map, the elution site inRP-HPLC of the peak VII-2 (D-2 in FIG. 16) completely agreed with thestandard product GN¹M3FX, β-D-GlcNAc-(1→2)-α-D-Man-(1→6) [α-D-Man-(1→3)][β-D-Xyl-(1→2)] β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-[α-L-Fuc-(1-3)]GlcNAc-PA(D-1 in FIG. 16).

From these data, the peak VII-2 was, as shown in FIG. 15B,β-D-GlcNAc-(1→2)-α-D-Man-(1→6) [α-D-Man-(1→3)] [β-D-Xyl-(1→2)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-[α-L-Fuc-(1→3)]GlcNAc-PA (GN¹M3FX).

The molecular weight (m/z 1324.0) of the peaks VII-1 and VIII-3 shown inFIG. 14B agreed with the calculated value of GlcNAcMan₃XylGlcNAc₂-PA(GNM3FX: 1324.24). Also, as a result of SF-HPLC analysis and IS-MS/MSanalysis, the N-acetylglucosaminidase digestion product of the peakVIII-1 agreed with Man₃XylGlcNAc₂-PA (M3X: 1121.05) (B in FIG. 18A).Furthermore, after the digestion by α-mannosidase, the SF-HPLC analysisand IC-MS/MS analysis revealed that the product agreed withManXylGlcNAc₂-PA (MX: 796.77) (C in FIG. 18A). As the structure ofGNM3X, two isomer types are considered, that is, α-D-Man-(1→6)[β-D-GlcNAc-(1→2)-α-D-Man-(1→3)] [β-D-Xyl-(1→2)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GN₁M3X) andβ-D-GlcNAc-(1→2)-α-D-Man-(1→6) [α-D-Man-(1→3)] [β-D-Xyl-(1→2)]β-D-Man-(1β4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GN¹M3X). On the ODS column, ithas been reported that GN₁M3X elutes in advance of GN¹M3X. When theelution sites of the peaks VII-1 and VIII-3 are considered, as shown inFIG. 15B, the peak VII-1 was α-D-Man-(1→6)[β-D-GlcNAc-(1→2)-α-D-Man-(1→3)][β-D-Xyl-(1→2)]β-D-Man-(1→4)-β-D-GlcNac-(1→4)-GlcNac-PA (GN₁M3X) and the peak VIII-3was β-D-GlcNAc-(1→2)-α-D-Man-(1→6) [α-D-Man-(1→6)] [β-D-Xyl-(1→2)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GN¹M3X).

The elution site of the peak X-1 shown in FIG. 14B in HPLC completelyagreed with the standard GN2M3 (1395.32) on the two-dimensional sugarchain map. Also, as a result of SF-HPLC analysis and IS-MS/MS analysis,the N-acetylglucosaminidase digestion product of sugar chain of the peakagreed with Man₃GlcNAc₂-PA (M3: 988.94). Furthermore, after thedigestion by α-mannosidase, the SF-HPLC analysis and IC-MS/MS analysisrevealed that the product agreed with ManGlcNAc₂-PA (M1: 664.66) (thedata are not shown). From these results, the peak X-1 was, as shown inFIGS. 15A and 15B, β-D-GlcNAc-(1→2)-α-D-Man-(1→6)[β-D-GlcNAc-(1→2)-α-D-Man-(1→3)]β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GN2M3).

The molecular weight (m/z 1529.5) of the peak X-2 shown in FIG. 14Bagreed with the calculated value of GlcNAc₂Man₃XylGlcNAc₂-PA (GN2M3FX:1527.43). Also, the elution site in HPLC completely agreed with the M3FXstandard product on the two-dimensional sugar chain map. Furthermore,the elution site of the α-mannosidase digestion product of sugar chainof this peak was not changed by the HPLC analysis. However, as a resultof the SF-HPLC analysis and IC-MS/MS analysis, theN-acetylglucosaminidase digestion product agreed with Man₂XylGlcNAc₂-PA(M3X: 1121.05) (B in FIG. 18B). After further digestion byα-mannosidase, the SF-HPLC analysis and IC-MS/MS analysis revealed thatthe product agreed with ManXylGlcNAc₂-PA (MX: 796.77) (C in FIG. 18A).From these data, the peak X-2 was, as shown in FIG. 15B,β-D-GlcNAc-(1→2)-α-D-Man-(1→6) [β-D-GlcNAc-(1→2)-α-D-Man-(1→3)][β-D-Xyl-(1→2)] β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-GlcNAc-PA (GN2M3X).

Other peaks IV-2, IV-3, V-2, V-3, V-5, VI-1, VI-2, VI-3, VI-4, VIII-2and IX shown in FIG. 14B were not an N-linked sugar chain, because as aresult of the IS-MS/MS analysis, signals agreeing with m/z 299.33(GlcNAc-PA) and m/z 502.52 (GlcNAc₂-PA) were not obtained.

4. Secretion of Horse Radish Peroxidase (HRP)

A foreign gene, horseradish peroxidase gene, which was obtained from35S-Cla (Kawaoka et al., J. Ferment. Bioeng., 78, 49-53 (1994)), wasinserted at Hind III and SacI site of vector pBI101 HmB (Akama et al.,Plant Cell Rep. 12, 7-11(1992)) and introduced into GT6 Strain. Aftercultivating the obtained clones of GT6 Strain (GT-HRP-5, GT-HRP-19), thesupernatant was collected, and subjected to a standard isoelectricfocusing electrophoresis. As a result, as indicated in FIG. 19,electrophoresis band of pI 7.8 of HRP was detected in the supernatant ofclones GT-HRP-5, GT-HRP-19, and thus confirmed that an foreign proteinwas also secreted from the plant cell transformed with the GalT gene.Further, FIG. 20 indicates the results of lectin staining of secretedproteins by clone GT-HRP-5, separated by standard SDS-PAGEelectrophoresis as described above. RCA120 staining indicates thatGT-HRP-5 has a positive signal (FIG. 20( c)), and thus it was indicatedthat secreted HRP had a galactose added sugar chain structure.

INDUSTRIAL APPLICABILITY

According to the present invention, a method for the secretoryproduction of a heterologous glycoprotein having a human-type sugarchain using a plant cell, a plant cell which can secrete thisglycoprotein, and a glycoprotein having a human-type sugar chain whichis secreted by a plant cell. The glycoprotein of the present inventionhas a human-type sugar chain and therefore, is free of antigenicity andis useful for the administration to animals including human.

1. A method for the secretory production of a glycoprotein having ahuman-type sugar chain from a plant cell, the method comprising: a)introducing into said plant cell a first heterologous nucleic acidsequence encoding an enzyme that is capable of performing a transferreaction of a galactose residue to a non-reducing terminalacetylglucosamine reside; b) introducing into said plant cell a secondnucleic acid sequence encoding a heterologous glycoprotein; and, c)culturing said plant cell such that the heterologous glycoprotein issecreted from said plant cell.
 2. The method of claim 1, wherein saidheterologous glycoprotein comprises a core sugar chain and an outersugar chain, said core sugar chain comprises a plurality of mannose andacetylglucosamines, and said outer sugar chain comprises a terminalsugar chain moiety containing a non-reducing terminal galactose.
 3. Themethod of claim 2 wherein said outer sugar chain has a linear structure.4. The method of claim 2 wherein said outer sugar chain has a branchedstructure.
 5. The method of claim 4 wherein said branched outer sugarchain is a mono-, bi, tri, or tetra-structure.
 6. The method of claim 1wherein said glycoprotein is free of fucose or xylose.
 7. The method ofclaim 1 further comprising a step of recovering the secretedglycoprotein.
 8. A transgenic plant cell comprising a first introducednucleic acid sequence encoding an enzyme capable of performing atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine and a second introduced nucleic acid sequence encodinga heterologous glycoprotein, wherein said enzyme adds a galactoseresidue to a glycoprotein comprising a core sugar chain and an outersugar chain, said core sugar chain substantially comprises a pluralityof mannoses and acetylglucosamines, and said outer sugar chain has aterminal sugar chain moiety containing a non-reducing terminal galactoseand wherein said transgenic plant cell secretes said heterologousglycoprotein.
 9. A transgenic plant cell according to claim 8 whereinsaid heterologous glycoprotein comprises a galactose residue.
 10. Atransgenic plant cell according to claim 8 wherein said heterologousglycoprotein is free of β1,2 xylose or α1,3 fucose.
 11. A transgenicplant cell according to claim 8 wherein said heterologous glycoproteincomprises a galactose residue added to an N-linked sugar of(N-acetylglucosamine)₁₋₂(Mannose)₂₋₅(N-acetylglucosamine)₂ selected fromthe group consisting of GlcNAc₁Man₃GlcNAc₂, GlcNAc₁Man₅GlcNAc₂,GlcNAc₂Man₃GlcNAc₂, and GlcNAc₁Man₄GlcNAc₂.
 12. A whole transgenic plantregenerated from the plant cell of claim 8 wherein said plant comprisessaid nucleic acid sequence encoding an enzyme capable of performing atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine.
 13. A transgenic seed produced by the plant of claim12 wherein said seed comprises said nucleic acid sequence encoding anenzyme capable of performing a transfer reaction of a galactose residueto a non-reducing terminal acetylglucosamine.